Creation of a Fully Functional Cysteine-Less Variant of Osmosensor and Proton-Osmoprotectant Symporter ProP from Escherichia coli and Its Application to Assess the Transporter's Membrane Orient

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• 作者：Doreen E. Culham ; Alexander Hillar ; James Henderson ; Anh Ly ; Yaroslava I. Vernikovska ; Kathleen I. Racher ; Joan M. Boggs ; and Janet M. Wood
• 刊名：Biochemistry
• 出版年：2003
• 出版时间：October 14, 2003
• 年：2003
• 卷：42
• 期：40
• 页码：11815 - 11823
• 全文大小：208K
• 年卷期：v.42,no.40(October 14, 2003)
• ISSN：1520-4995

文摘

Transporter ProP of Escherichia coli is an osmosensor and an osmoprotectant transporter.Previous results suggest that medium osmolality determines the proportions of ProP in active and inactiveconformations. A cysteine-less (Cys-less) variant was created and characterized as a basis for structuraland functional analyses based on site-directed Cys substitution and chemical labeling of ProP. Parametersdescribing the osmosensory and osmoprotectant transport activities of Cys-less ProP-(His)₆ variants wereexamined, including the threshold for osmotic activation and the absolute transporter activity at highosmolality (in both cells and proteoliposomes), the dependence of K_M and V_max for proline uptake onosmolality, and the rate constant for transporter activation in response to an osmotic upshift (in cellsonly). Variant ProP-(His)₆-C112A-C133A-C264V-C367A (designated ProP*) retained similar activitiesto ProP-(His)₆ in both cells and proteoliposomes. The bulky Val residue was favored over Ala or Ser atposition 264, whereas Val strongly impaired function when placed at position 367, highlighting theimportance of residues at those positions for osmosensing. In the ProP* background, variants with asingle Cys residue at positions 112, 133, 241, 264, 293, or 367 retained full function. The native Cys atpositions 112, 133, 264, and 367, predicted to be within transmembrane segments of ProP, were poorlyreactive with membrane-impermeant thiol reagents. The reactivities of Cys at positions 241 and 293 wereconsistent with exposure of those residues on the cytoplasmic and periplasmic surfaces of the cytoplasmicmembrane, respectively. These observations are consistent with the topology and orientation of ProPpredicted by hydropathy analysis.